Composition of matter and process

ABSTRACT

Novel compounds prepared by microbial transformation using novel mutants to selectively degrade steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive. These compounds can be used as intermediates to make useful steroids.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studiedand documented. Apparently, the earliest such work was by Mamoli andVercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed thereduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast.More recently, Peterson and Murray disclosed the 11α-hydroxylation ofprogesterone with the fungus Rhizopus nigricans; see, U.S. Pat. No.2,602,769 (1952). Also recently, Kraychy et al. in U.S. Pat. No.3,684,657 (1972) discloses the selective microbiological degradation ofsteroidal 17-alkyls by fermenting a steroid containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 toprepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck etal. in U.S. Pat. No. 3,759,791 (1973) disclose the selectivemicrobiological preparation of androst-4-ene-3,17-dione by fermenting asteroid of the cholestane or stigmastane series containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805.

The open ring form of compound 1,7α-methyl-perhydroindanedione-(1,5)-[β-propyl alcohol-(4)], has beendisclosed by Schubert et al. in 1964 (Steroids 4: 581-586) as anintermediate in the degradation of progesterone by Mycobacteriumsmegmatis.

BRIEF SUMMARY OF THE INVENTION

Mutants which are characterized by their ability to selectively degradesteroids with or without 17-alkyl side chains of from 2 to 10 carbonatoms, inclusive, and accumulate predominantly the following compoundsin the fermentation beer:

3Aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandione hemiketal##STR1## hereinafter referred to as Compound I; and

3Aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone##STR2## hereinafter referred to as Compound IV.

These mutants can be obtained from sterol-degrading microorganisms ofthe following genera by using the mutation procedures disclosed hereinor other mutation procedures: Arthrobacter, Bacillus, Brevibacterium,Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, andStreptomyces. A preferred genus is Mycobacterium. Exemplary species ofthis genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M.fortuitum, and M. butyricum. Specifically exemplified herein is a novelmutant microorganism, Mycobacterium fortuitum, NRRL B-8129, which isused to selectively degrade steroids with or without 17-alkyl chainscontaining from 2 to 10 carbon atoms, inclusive, to compounds I and IV.Examples of suitable steroids are sitosterols, cholesterol,stigmasterol, campesterol, and like steroids with 17-alkyl side chainsof from 2 to 10 carbon atoms, inclusive, and androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.These steroid substrates can be in either the pure or crude form.

DETAILED DESCRIPTION OF THE INVENTION The Microorganisms

Mutants which are characterized by their ability to selectively degradesteroids with or without 17-alkyl side chains containing from 2 to 10carbon atoms, inclusive, and accumulate compounds I and IV in thefermentation beer can be obtained by mutating sterol-degradingmicroorganisms of the following genera: Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Mycobacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces. Mycobacterium fortuitum,ATCC 6842, has been mutated, as disclosed herein, to give a novellaboratory mutant microorganism. The 1974 ATCC Catalogue discloses thefollowing alongside the listing of ATCC 6842: "J.C. Cruz 2. Coldabscess. Acta Med. Rio de Janeiro 1:1 (1936). Medium 90 37C". M.fortuitum, ATCC 6842, degrades sterols non-selectively to smallmolecular weight compounds, e.g., CO₂ +H₂ O. Thus, this microorganism isnot suitable as a selective steroid degrader.

Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resultedin the production of a novel mutant which selectively degrades steroidswith or without 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, to produce compounds I and IV predominantly in thefermentation beer. This mutant microorganism of M. fortuitum has beengiven the accession number NRRL B-8129, by the Northern RegionalResearch Laboratory, U.S. Department of Agriculture, Peoria, Ill.,U.S.A., where it has been deposited in the permanent collection. Asubculture of this microorganism is freely available from thisdepository by request made thereto. It should be understood that theavailability of the culture does not constitute a license to practicethe subject invention in derogation of patent rights granted with thesubject instrument by governmental action.

The morphology and drug sensitivities of M. fortuitum, NRRL B-8129, areindistinguishable from that of the parent M. fortuitum, ATCC 6842. BothM. fortuitum cultures are acid-fast non-motile, non-spore-formingbacilli belonging to the family Mycobacteriaceae of the orderActinomycetales. According to Runyon's classification (Runyon, E. H.1959 Med. Clin. North America 43: 273) it is a nonchromogenic group IVmycobacterium, i.e., it grows rapidly at low temperature to producenonpigmented colonies on relatively simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8129, are clearlydistinguishable in their action on steroid molecules. As disclosedabove, M. fortuitum ATCC 6842 is a degrader of steroids, whereas M.fortuitum NRRL B-8129 is a selective degrader. This property of M.fortuitum NRRL B-8129 makes it highly useful, as disclosed herein.

The mutation of M. fortuitum ATCC 6842 to give M. fortuitum NRRL B-8129was accomplished by the use of nitrosoguanidine. The details of theprocedure are described infra. Though mutation procedures are generallyknown in the art, there is no known art which teaches or even suggeststhe type of mutants, if any, which might be obtained by use of thesubject mutation procedure. Also, though the mutation and transformationprocedures, disclosed herein, are detailed for a Mycobacterium, itshould be understood that similar or equivalent procedures can be usedwith microorganisms of the other genera, as disclosed herein.

The Transformation Process

The selective transformation of the subject invention can be effected ina growing culture of M. fortuitum NRRL B-8129 by either adding theselected steroid substrate to the culture during the incubation period,or incorporating it in the nutrient medium prior to inoculation. Thesteroid can be added singly or in combination with another steroid. Thepreferred, but not limiting, range of concentration of the steroid inthe culture is about 0.1 to about 100 grams per liter. The culture isgrown in a nutrient medium containing a carbon source, for example, anassimilable carbohydrate, and a nitrogen source, for example, asassimilable nitrogen compound or proteinaceous material. Preferredcarbon sources include glucose, brown sugar, sucrose, glycerol, starch,cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogensources include cornsteep liquor, yeast, autolyzed brewer's yeast withmilk solids, soybean meal, cottonseed meal, cornmeal, milk solids,pancreatic digest of casein, fish meal, distiller's solids, animalpeptone liquors, meat and bone scraps, ammonium salts and the like.Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

The transformation process can range from about 72 hours to 15 days. Theincubation temperature during the transformation process can range fromabout 25° C. to about 37° C., with 30° C. being preferred. The contentsof the transformation vessel are aerated with sterilized air andagitated to facilitate growth of the microorganism, and, thus, enhancethe effectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thinlayer chromatography using silica gel plates (E. Merck, Darmstadt) and asolvent sytem consisting of 2:3 (by volume) ethyl acetate-cyclohexane,the desired transformed steroids are recovered by means well known inthe art. For example, the fermentation (transformation) reactionmixture, including the fermentation liquor and cells, can be extractedwith a water-immiscible organic solvent for steroids. Suitable solventsare methylene chloride (preferred), chloroform, carbon tetrachloride,ethylene chloride, trichloroethylene, ether, amyl acetate, benzene andthe like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtratevacuum distilled to dryness. The resulting residue containing thedesired transformed steroids then can be dissolved in 10% chloroform inSkellysolve B and chromatographed on silica gel, using Skellysolve B(isomeric hexanes) and mixtures thereof with increasing amounts of ethylacetate as developing solvent. This procedure elutes compound I.Compound IV can be eluted with, for example, 5% methanol in ethylacetate. Crystalline products can be obtained by use of a solvent, forexample, ethyl acetate. The solution then can be cooled to roomtemperature and filtered to remove precipitated steroid. The desiredtransformed steroids can also be obtained from the remaining supernatantupon evaporation of the solvent in the supernatant.

Compounds I and IV are useful as intermediates in the chemical synthesisof useful steroids. For example, the subject compounds can be convertedto starting material for the process disclosed in U.S. Pat. No.3,880,884 which discloses a process for the total synthesis of useful19-nor steroids. This conversion to starting material can be done byprocedures known in the art, e.g., by oxidation with chromic acid inacetic acid followed by treatment with acetic anhydride and sodiumacetate. See Biochem. 2: 1238-1243 and J.A.C.S. 85: 2135-2137.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NRRL B-8129 From M.fortuitum ATCC 6842.

a. Nitrosoguanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

    ______________________________________                                        Nutrient Broth (Difco)                                                                             8       g/liter                                          Yeast Extract        1       g/liter                                          Sodium Propionate    0.5     g/liter                                          Distilled Water, q.s.                                                                              1       liter                                            ______________________________________                                    

The pH is adjusted to 7.0 with 1N NaOH prior to sterilization at 121° C.for 20 minutes.

The cells are grown to a density of about 5 × 10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoguanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C. in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

    ______________________________________                                        NH.sub.4 NO.sub.3    1.0      g/liter                                         K.sub.2 HPO.sub.4    0.25     g/liter                                         MgSO.sub.4 . 7H.sub.2 O                                                                            0.25     g/liter                                         NaCl                 0.005    g/liter                                         FeSO.sub.4 . 7H.sub.2 O                                                                            0.001    g/liter                                         Distilled Water, q.s.                                                                              1        liter                                           ______________________________________                                    

The pH is adjusted to 7.0 with 1N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

b. Selection And Isolation Of Mutant M. fortuitum NRRL B-8129.

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

    ______________________________________                                        Glycerol             10.0    g/liter                                          Na.sub.2 HPO.sub.4   8.4     g/liter                                          KH.sub.2 PO.sub.4    4.5     g/liter                                          NH.sub.4 Cl          2.0     g/liter                                          MgSO.sub.4 . 7H.sub.2 O                                                                            0.3     g/liter                                          FeCl.sub.3 . 6H.sub.2 O                                                                            0.05    g/liter                                          Distilled Water, q.s.                                                                              1       liter                                            ______________________________________                                    

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced bythe mutagenesis procedure, e.g. cultures that require vitamins, growthfactors, etc. in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates autoclaved for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described by Peterson, G. E., H. L.Lewis and J. R. Davis. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3: 275-276. The minimal salts medium in these plates isas described above in section (a) of Example 1. Agar (15 g/liter), andan appropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri plates. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test platescontaining AD as the sole carbon source, are purified by streaking ontonutrient agar plates. After growth at 28° C., individual clones arepicked from the nutrient agar plates with sterile toothpicks andretested by inoculating grided plates containing AD as the carbonsource. Purified isolates which still exhibit a phenotype different fromthe parental culture are then evaluated in shake flasks.

c. Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

    ______________________________________                                        Glycerol             10.0    g/liter                                          Na.sub.2 HPO.sub.4   8.4     g/liter                                          KH.sub.2 PO.sub.4    4.5     g/liter                                          NH.sub.4 Cl          2.0     g/liter                                          MgSO.sub.4 . 7H.sub.2 O                                                                            0.3     g/liter                                          FeCl.sub.3 . 6H.sub.2 O                                                                            0.05    g/liter                                          Distilled Water, q.s.                                                                              1       liter                                            ______________________________________                                    

Soyflour (1 g/liter) is blended into the medium and then sitosterol (10g/liter) is also blended into the medium. After the flasks areautoclaved for 20 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

    ______________________________________                                        Nutrient Broth (Difco)                                                                              8 g/liter                                               Yeast Extract         1 g/liter                                               Glycerol              5 g/liter                                               Distilled Water, q.s. 1 liter                                                 ______________________________________                                    

The pH is adjusted to 7.0 with 1N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 ml of sterile transformation medium. Theflasks are then incubated at 28° to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride. Portions of theextracts are analyzed by thin layer chromatography using silica gel andthe solvent system described above, i.e., 2:3 (by volume) ethylacetate-cyclohexane, and by gas-liquid chromatography. Evidence of thepresence of compounds I and IV confirms the selective degradation ofsitosterol by the novel mutant produced from the parent M. fortuitumATCC 6842.

EXAMPLE 2

Transformation of Sitosterol To Compounds I and IV.

The medium used is the same as in Example 1c. This medium is sterilizedby heating 30 minutes at 121° C., whereupon it is cooled to 30° C. andthen inoculated with 10 parts of a seed culture of the mutantmycobacterium M. fortuitum NRRL B-8129, prepared as described in Example1 (c). The inoculated mixture is incubated at 30° C. for 336 hours withagitation to promote submerged growth. Following incubation, the mixtureis extracted with methylene chloride. The extract is filtered throughdiatomaceous earth and the filtrate is vacuum distilled to dryness. Theresidue is taken up in 10% chloroform in Skellysolve B andchromatographed on silica gel, using Skellysolve B and mixtures thereofwith increasing amounts of ethyl acetate as developing solvent. Thisprocedure elutes compound I. Compound IV is eluted from the column with5% methanol in ethyl acetate. The order of elution of the compounds is I→ IV. The R_(f) values for these compounds on thin layer chromatography(tlc) is as follows (cyclohexane-ethyl acetate; 3:2):

I = 0.37

iv = 0.05

appropriate fractions, as determined by tlc, are pooled and evaporatedto dryness to afford good yields of compounds I and IV.

Upon recrystallization from ethyl acetate, compound I, a 1:1 mixture oftwo epimers, melts at 100°-112° C. and compound IV melts at 148°-150° C.

Traces of3aα-H-4α-[3'-propanal]-5α-hydroxy-7aβ-methylhexahydro-1-indanonehemiacetal and 3aα-H-4α-[3'-propionicacid]-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone were shown bythin layer chromatography to be present.

EXAMPLE 3

By substituting cholesterol for sitosterol in Example 2 there isobtained compounds I and IV.

EXAMPLE 4

By substituting stigmasterol in Example 2 for sitosterol there isobtained compounds I and IV.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there isobtained compounds I and IV.

EXAMPLE 6

By adding a combination of any of the steroids in Examples 2-5, inaddition to sitosterol, or in place of sitosterol, in Example 2 there isobtained compounds I and IV.

EXAMPLE 7

By substituting a sterol-degrading microorganism from the generaArthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842 there are obtained mutantmicroorganisms which are characterized by their ability to selectivelydegrade steroids with or without a 17-alkyl side chain of from 2 to 10carbon atoms, inclusive, and accumulate compounds I and IV in thefermentation beer.

EXAMPLE 8

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8129 in Examples 2-6, there is obtained compounds I and IV.

EXAMPLE 9

By substituting a sterol-degrading microorganism selected from the groupconsisting of Mycobacterium phlei, M. smegmatis, M. rhodochrous, M.mucosum, and M. butyricum for M. fortuitum ATCC 6842 in Example 1 thereare obtained mutant microorganisms which are characterized by theirability to selectively degrade steroids with or without a 17-alkyl sidechain of from 2 to 10 carbon atoms, inclusive, and accumulate compoundsI and IV in the fermentation beer.

EXAMPLE 10

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8129 in Examples 2-6, there is obtained compounds I and IV.

EXAMPLE 11

By substituting a compound selected from the group consisting ofandrost-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone for sitosterol in Example 2there is obtained compounds I and IV.

Example 12

By substituting a combination of two or more compounds selected from thegroup consisting of sitosterol, cholesterol, stigmasterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone for sitosterol in Example 2there is obtained compounds I and IV.

EXAMPLE 13

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8129 in Examples 11 and 12, there is obtained compounds I and IV.

EXAMPLE 14

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8129 in Examples 11 and 12, there is obtained compounds I and IV.

We claim:
 1. A process for preparing a compound of the formula ##STR3##which comprises cultivating Mycobacterium fortuitum NRRL B-8129 in anaqueous nutrient medium under aerobic conditions in the presence of asteroid with or without a 17-alkyl side chain containing from 2 to 10carbon atoms, inclusive.
 2. A process, according to claim 1, whereinsaid steroid is selected from the group consisting of sitosterol,cholesterol, stigmasterol, campesterol, androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.3. A process for preparing a compound, as defined in claim 1, whichcomprises cultivating Mycobacterium fortuitum NRRL B-8129 in an aqueousnutrient medium under aerobic conditions in the presence of a mixture oftwo or more steroids.
 4. A process, according to claim 3, wherein saidsteroid mixture is selected from the group consisting of sitosterol,cholesterol, stigmasterol, campesterol, androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.5. A process for preparing a compound, as defined in claim 1, whichcomprises cultivating a Mycobacterium mutant which is characterized byits ability to selectively degrade steroids with or without a 17-alkylside chain of from 2 to 10 carbon atoms, inclusive, and accumulatepredominantly 3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandionehemiketal and3aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone in thefermentation beer, in an aqueous nutrient medium under aerobicconditions in the presence of a steroid with or without a 17-alkyl sidechain containing from 2 to 10 carbon atoms, inclusive.
 6. A process,according to claim 5, wherein said mutant microorganism is cultivated inan aqueous nutrient medium under aerobic conditions in the presence of amixture of two or more steroids.
 7. A process, according to claim 5,wherein said steroid is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 8. A process, according toclaim 6, wherein said steroid mixture is selected from the groupconsisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 9. A process for preparing acompound of the formula ##STR4## which comprises cultivatingMycobacterium fortuitum NRRL B-8129 in an aqueous nutrient medium underaerobic conditions in the presence of a steroid with or without a17-alkyl side chain containing from 2 to 10 carbon atoms, inclusive. 10.A process, according to claim 9, wherein said steroid is selected fromthe group consisting of sitosterol, cholesterol, stigmasterol,campesterol, androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 11. A process for preparing acompound, as defined in claim 9, which comprises cultivatingMycobacterium fortuitum NRRL B-8129 in an aqueous nutrient medium underaerobic conditions in the presence of a mixture of two or more steroids.12. A process, according to claim 11, wherein said steroid mixture isselected from the group consisting of sitosterol, cholesterol,stigmasterol, campesterol, androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.13. A process for preparing a compound, as defined in claim 9, whichcomprises cultivating a Mycobacterium mutant which is characterized byits ability to selectively degrade steroids with or without a 17-alkylside chain of from 2 to 10 carbon atoms, inclusive, and accumulatepredominantly 3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandionehemiketal and3aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone in thefermentation beer, in an aqueous nutrient medium under aerobicconditions in the presence of a steroid with or without a 17-alkyl sidechain containing from 2 to 10 carbon atoms, inclusive.
 14. A process,according to claim 13, wherein said mutant microorganism is cultivatedin an aqueous nutrient medium under aerobic conditions in the presenceof a mixture of two or more steroids.
 15. A process, according to claim13, wherein said steroid is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 16. A process, according toclaim 14, wherein said steroid mixture is selected from the groupconsisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 17. A process for preparing afermentation beer containing compounds of the formulae ##STR5## whichcomprises cultivating Mycobacterium fortuitum NRRL B-8129 in an aqueousmedium under aerobic conditions in the presence of a steroid with orwithout a 17-alkyl side chain containing from 2 to 10 carbon atoms,inclusive.
 18. A process, according to claim 17, wherein said steroid isselected from the group consisting of sitosterol, cholesterol,stigmasterol, campesterol, androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.19. A process for preparing a fermentation beer, as defined in claim 17,which comprises cultivating Mycobacterium fortuitum NRRL B-8129 in anaqueous nutrient medium under aerobic conditions in the presence of amixture of two or more steroids.
 20. A process, according to claim 19,wherein said steroid mixture is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 21. A process for preparing afermentation beer, as defined in claim 17, which comprises cultivating aMycobacterium mutant which is characterized by its ability toselectively degrade steroids with or without a 17-alkyl side chain offrom 2 to 10 carbon atoms, inclusive, and accumulate predominantly3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandione hemiketal and3aα-H-4α-[3'-propanol] -5'α-hydroxy-7aβ-methylhexahydro-1-indanone inthe fermentation beer, in an aqueous nutrient medium under aerobicconditions in the presence of a steroid with or without a 17-alkyl sidechain containing from 2 to 10 carbon atoms, inclusive.
 22. A process,according to claim 21, wherein said mutant microorganism is cultivatedin an aqueous nutrient medium under aerobic conditions in the presenceof a mixture of two or more steroids.
 23. A process, according to claim21, wherein said steroid is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.
 24. A process, according toclaim 22, wherein said steroid mixture is selected from the groupconsisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone.